Birth defects, i.e. abnormalities developing in fetal life and present at birth, is the major cause of infant death, defined as death within a year of birth, in the USA. Congenital heart defects occur with a frequency of 8-9 cases per 1,000 live births. CHD is the most common group of severe birth defects and is the most costly in terms of hospitalization. Up to 25% of cases with major CHD in newborns are not diagnosed prior to discharge from the hospital.
Congenital aortic valve stenosis (AVS), defined as incomplete obstruction of the valve orifice, is an important category of structural heart defect, and occurs in 3-6% of such cases. There is variability in both the site of obstruction and severity of the obstruction. Sites of obstruction are sub-classified as valvular, subvalvular and supravalvular. About half of infants with severe AVS require surgery. Mild aortic stenosis is difficult to detect in prenatal life, however critical aortic stenosis can lead to left ventricular myocardial dysfunction with endocardial fibroelastosis, left atrial dilation and narrowing of the aortic root. These changes can be a prelude to the development of hypoplastic left heart syndrome.
Based on the high percentage of major CHD that fail to be diagnosed in newborns, it has been recommended that measurement and monitoring of tissue oxygen levels “pulse oximetry” be performed in all newborns to detect low tissue oxygen levels which may be a sign of the presence of a major CHD. There is a clear need to develop screening tests and other markers for the accurate prediction of CHD in the general population both in newborns and also in later stages of postnatal life.
Heart development in embryonic and fetal life requires the coordination and orchestration of a large number of different genes. A relatively small percentage of CHD cases is known to be related to gene mutations which are changes in the normal sequence in which the basic building block (“nucleotides”) are arranged in the DNA of the gene. Such mutations lead to malfunctioning or nonfunctioning of genes (i.e. altered amounts, of or the production of abnormal types of proteins) that are important for normal heart development.
In the last six decades an important mechanism for controlling gene function called “epigenetics” has been discovered and extensively investigated. Epigenetics is defined as heritable (i.e. passed onto offspring) changes in gene expression that are not due to mutations i.e. changes in the sequence of, loss or gain of nucleotides in the gene. Rather, epigenetics is a reversible regulation of gene expression by several other potential mechanisms. One such mechanism which is currently the most extensively studied is DNA methylation. Other mechanisms include: changes on the 3 dimensional structure of the DNA, histone protein modification or micro-RNA inhibitory activity.
Cytosine methylation is chemically stable and can be measured in DNA from any source including fresh, stored or archived tissues such as DNA preserved in pathology slides or formalin-fixed paraffin blocks. In addition DNA released from destroyed cells and present in body fluids, cfDNA, can also be a tested for cytosine methylation.
The methylation of cytosine nucleotides within a gene, particularly in the promoter region (which controls gene expression) of said gene is known to be a mechanism of controlling overall gene activity. Classically, the methylation of cytosine is associated with inhibition of gene transcription. However, in certain genes, methylation of cytosine is known to have the reverse effect i.e. promotion of gene transcription.
Commonly used techniques for measuring cytosine methylation include but are not limited to bisulfite-based methylation assay. The addition of bisulfite to DNA results in the conversion of unmethylated cytosine results in the methylation of the cytosine (i.e. addition of an extra carbon atom to position #5 of the hexagonal ring structure of the cytosine nucleotide) and its ultimate conversion to the nucleotide uracil. Uracil has similar binding properties to thiamine in the DNA sequence. Previously methylated cytosine does not undergo this chemical conversion on exposure to bisulfate. Bisulfite assays can thus be used to discriminate previously methylated versus unmethylated cytosine.
Thus the methylation status of cytosine throughout the DNA can be said to indicate the relative expression status of multiple genes throughout the genome. The technique therefore permits simultaneous analysis of the relative level of activation of multiple genes directly or indirectly involved in cardiac development since the mechanism of action of external substances and influences on the cell is largely through their effect on gene function, genome wide DNA methylation also represents the integrated effect of a large number of external (prenatal alcohol and tobacco exposure, anti-folate metabolites etc.) and internal influences on the numerous genes involved in cardiac development. Overall therefore, the differences in cytosine methylation in CHD and normal groups can be used to estimate the risk of and predict the likelihood of CHD in an individual by comparing their cytosine methylation levels to appropriate reference standards.
Despite the frequency and importance of CHD, there is no laboratory test for the routine population screening of embryos, fetuses, newborns or in later stages of post-natal life for CHD. There is a significant need for screening tests that will facilitate the early identification of, medical surveillance of, and treatment of newborns and other individuals with CHDs.